PROJECT SUMMARY Because it is infectious, deadly, and difficult to treat, Naegleria fowleri requires strict laboratory containment, making scientific progress slow and costly, and leaving the organism largely unstudied. The related yet nonin- fectious Naegleria gruberi shares key genes and behaviors thought to drive Naegleria fowleri pathogenesis, including putative virulence factors and rapid destruction and ingestion of brain tissue that results in patient symptoms and death. The applicants are therefore developing N. gruberi as a model to understand the genes and mechanisms driving N. fowleri pathogenesis. This application seeks to develop two independent methods for manipulating N. gruberi gene function: (Aim 1) RNA-interference (RNAi): Naegleria normally eat bacteria, and the Naegleria genome encodes RNAi machinery. RNAi methods developed for C. elegans and ciliates will be adapted by growing Naegleria amoebae on lawns of bacteria expressing double-stranded RNA to induce cleavage of target mRNAs by the native Naegleria dicer. (Aim 2) Gene disruption by CRISPR. This technology has been used for specific and efficient genome engineering of diverse protist pathogens. A highly efficient N. gruberi electroporation protocol will be adapted for delivery of purified CAS9 holoenzyme into live cells. Both approaches will target three genes as proof-of-platform, each of which is used specifically to build flagella upon starvation. Disruption of any of these genes should result in cells lacking flagella, an easily quantified and non- lethal phenotype. The status quo as it pertains to understanding N. fowleri pathogenesis is to use correlative approaches, particularly comparing infectious N. fowleri to less virulent strains and nonpathogenic species. Although this approach has revealed differences in heat tolerance and expression levels of individual genes, by definition it excludes pathogenic behaviors that are shared with noninfectious species, including destruction of brain tissue, the leading direct cause of patient symptoms and death. The proposed research is innovative, in the applicant's opinion, because it represents a substantive departure from the status quo by allowing direct testing of potential molecular mechanisms driving host tissue destruction. The ability to interfere with Naegleria gruberi gene function will allow the first direct testing of the roles of putative virulence factors in the molecular mechanisms driving pathogenic behaviors.